Reconstitution of Saccharomyces cerevisiae prereplicative complex assembly in vitro

The assembly of the prereplicative complex (pre‐RC) at the origin of replication in eukaryotes is a highly regulated and highly conserved process that plays a critical role in preventing multiple rounds of DNA replication per cell division cycle. This study analyzes the molecular dynamics of the assembly of Saccharomyces cerevisiae pre‐RC in vitro using ARS1 plasmid DNA and yeast whole cell extracts. In addition, pre‐RC assembly was reconstituted in vitro using ARS1 DNA and purified origin‐recognition complex (ORC), Cdc6p and Cdt1p‐Mcm2‐7p. The results reveal sequential recruitment of ORC, Cdc6p, Cdt1p and Mcm2‐7p on to ARS1 DNA. When Mcm2‐7p is maximally loaded, Cdc6p and Cdt1p are released, suggesting that these two proteins are co‐ordinately regulated during pre‐RC assembly. In extracts from sid2‐21 mutant cells that are deficient in CDT1, ORC and Cdc6p bind to ARS1 but Cdt1p and Mcm2‐7p do not. However, Mcm2‐7p does bind in the presence of exogenous Cdt1p or Cdt1p‐Mcm2‐7p complex. Cdt1p‐Mcm2‐7p complex, which was purified from G1‐, early S or G2/M‐arrested cells, exhibits structure‐specific DNA binding, interacting only with bubble‐ or Y‐shape‐DNA, but the biological significance of this observation is not yet known.